LC-MSN METHOD FOR QUALITATIVE & QUANTITATIVE ANALYSIS OF COMPLEX LIPID MIXTURES

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 虽然纳米质谱仪是表征简单脂类混合物(1，2)的一个很好的选择，但对于高度复杂的样品的定性和定量分析往往是不够的。所描述的大多数分离方法都是有限的，因为它们要么只针对感兴趣的特定类别(3)，要么不适合于MS，这是一种更好的检测方法，特别是对于少量样品的分析。我们开发了一种简单、可重现的三步法，通过采用文献中描述的分离系统来进行脂类分析(4，5)。经过可选的初始分级后，正相高效液相色谱-质谱仪首先提供分类分离，然后反相LC-MS/MS系统回答剩余的问题。 方法：(A)将分离提取的低密度脂蛋白和脂质标准品逐级传递到硅胶60树脂上，用甲基叔丁基醚(MTBE)洗脱，然后用甲醇洗脱。(B)这两个组分或全部样品(或一套标准)在Waters/YMC微孔PVA-Sil高效液相色谱柱上进一步分离，并以正离子和负离子模式进行质谱分析。使用两种不同的梯度，一种基于正庚烷和MTBE，另一种基于MTBE和在甲酸铵存在下的甲醇，分别用于分离更多的非极性和更多的极性脂肪。量化是基于这一步的。前者需要柱后馈电才能进行适当的电离。(C)可使用Waters CapLC系统上的C18 Atlantis毛细管柱，通过与三重四极杆、QoTOF MS或LTQ-Orbitrap MS接口的反相LC-MS/MS，或通过纳米喷雾MS/MS和/或前体离子扫描来进一步表征所获得的馏分。 含有多种非极性、磷脂和糖脂的脂类和糖脂标准品通过MTBE和甲醇从硅胶60树脂中洗脱，可根据极性重复分离。当用于生物样品时，这一步骤也用于保护下面的柱，但并不总是必要的。样品在PVA-Sil正相柱上使用两种不同的梯度分离，一种用于非极性脂类的测定，另一种用于极性脂类的测定。正常相柱上的这些分离允许至少半定量检测。量化的准确性在很大程度上取决于可用的内部和外部标准的质量。 收集的馏分用纳米级联用质谱(MS/MS、前驱体离子扫描和中性损失扫描)进行部分研究，以确定存在的分子物种。在反相柱上实现了分子物种的干净分离。特别是低丰度的PES可以通过这种方式得到确认。 LCMS方法提供了一种相当健壮且技术简单的方法 复杂脂类混合物的研究。我们已经将该方法应用于与全长和截短的载脂蛋白相关的脂类的分析，在一个正常个体和一个具有导致截短蛋白产生的基因修饰的个体中。我们发现，这两种情况下的脂质模式是不同的。这些结果已经发表(7)，并引起了极大的兴趣，从美国和其他地方的研究人员开始在自己的实验室实施这种方法时联系我们的数量来看。墨菲等人。最近发表了对该方法的修改，简化了提取和层析，但保留了磷脂。(8) 1)M.Puffer和R.C.Murphy(2003)。质谱学评论22,332-。 2)X.Han.和R.W.Gross(2005)。质谱学版本24,367-412。 3)R.C.墨菲等人。(2001年)。化学。第101版，479-526号。 4)J.Hamilton和K.Comai(1988)。脂23、1046-49和1150-53。 5)W.W.克里斯蒂等人。(1995年)。J.高分辨率。变色剂。18，97-100。 6)F.K.韦尔蒂等人。(1991年)。J·克莱恩。投资。87年，1748-1754年。 7)U·Sommer等人。(2006)J.Lipid Res.47,804-814. 8)P.M.Hutchins等人。(2008)J.Lipid Res.49,804-813。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. While nanospray MS is a good choice for the characterization of simple lipid mixtures (1,2), it is often not sufficient for the qualitative and quantitative analysis of highly complex samples. Most separation methods described are limited, in that they either target only specific classes of interest (3), or are not well suited for MS, the superior detection method, especially for analyses of small amounts of samples. We have developed a simple, reproducible three-step method for lipid analysis by adapting separation systems described in the literature for the chromatography of lipids (4,5). After an optional initial fractionation, normal phase HPLC-MS first provides class separation and then a reversed phase LC-MS/MS system answers remaining questions. Methods: (a) Isolated and extracted LDL lipids and lipid standards are passed stepwise onto and eluted off Silica 60 resin with MTBE (methyl t-butyl ether), followed by methanol. (b) Either these two fractions or the full sample (or set of standards) are further separated on a Waters/YMC microbore PVA-Sil HPLC column and are detected by mass spectrometry in positive and negative ion modes. Two different gradients are used, one based on heptane and MTBE, and one based on MTBE and methanol in the presence of ammonium formate, for the separation of more nonpolar and more polar lipids, respectively. Quantification is based on this step. The former requires a postcolumn feed for proper ionization. (c) Fractions obtained can be further characterized by reversed phase LC-MS/MS using a C18 Atlantis capillary column on a Waters CapLC system interfaced to the triple quadrupole, QoTOF MS, or LTQ-Orbitrap MS, or by nanospray MS/MS and/or precursor ion scanning. Lipid and glycolipid standards containing diverse nonpolar, phospho- and glycolipids have been reproducibly separated on the basis of polarity by elution from Silica 60 resin with MTBE and methanol. This step, when used for biological samples, also serves to protect the following column, but is not always necessary. The sample is separated on a PVA-Sil normal phase column using two different gradients, one for determination of nonpolar lipids, and the other for polar lipids. These separations on the normal phase column allow for an at least semi-quantitative detection. The accuracy of the quantification depends mostly on the quality of internal and external standards available. The collected fractions are partially investigated by nanospray MS (MS/MS, precursor ion scanning and neutral loss scanning) for the determination of the molecular species present. A clean separation of molecular species has been achieved on a reversed phase column. Especially the low abundant PEs can be confirmed that way. The LCMS methodology provides a fairly robust and technically simple method for the investigation of complex lipid mixtures. We have applied the method to the analysis of lipids associated with full-length and truncated apolipiprotein in a normal indivicual and one who has a genetic modification that results in production of the truncated protein. We have found that the lipid pattern is different in the two cases. these results have been published (7) and have drawn significant interest, judging from the number of investigators from the US and elsewhere who have contacted us about this approach as they begin to implement it in their own laboratories. Murphy et al. recently published a modification to the method that simplifies the extraction and chromatography but leaves behind the phospholipids. (8) 1) M. Puffer and R.C. Murphy (2003). Mass Spectrometry Reviews 22, 332-64. 2) X. Han and R.W. Gross (2005). Mass Spectrom Rev. 24, 367-412. 3) R.C. Murphy et al. (2001). Chem. Rev. 101, 479-526. 4) J. Hamilton, and K. Comai (1988). Lipids 23, 1046-49 & 1150-53. 5) W.W. Christie et al. (1995). J. High Resol. Chromatogr. 18, 97-100. 6) F.K. Welty et al. (1991). J. Clin. Invest. 87, 1748-1754. 7) U. Sommer et al. (2006) J. Lipid Res. 47, 804-814. 8) P. M. Hutchins et al. (2008) J. Lipid Res. 49, 804-813.